This proposal focuses on identifying and diversifying novel monoclonal antibodies (Mabs) isolated from humans (huMabs) that are exposed to viral sexually transmitted pathogens. Coupling antibody discovery technology (FACS/RT-PCR) with a versatile and rapid (days to weeks) production system facilitates evaluation of the greatest range of protective huMabs. The variable regions isolated from STI exposed individuals when matched with an array of pre-built scaffolds that have defined constant region features (e.g. multimeric, + complement binding), significantly increases the diversity and utility of the STI Mab portfolio for understanding antibody correlates of protection for vaccines and microbicides. In the first Specific Aim, we will isolate and clone huMabs from STI exposed individuals. Initial efforts will focus on isolation of human Mabs from the antibody repertoire of HIV exposed uninfected (HIV-EU) individuals. In the later years of the project, efforts will be expanded to HSV-2 and HPV seropositive individuals, as well as individuals immunized in Project 3 with the experimental vaccines generated by the Production Core. Antigen-specific IgG+, IgM+, and IgA+ B cells from these individuals will be subjected to single ceil sorting (FACS) against antigens identified as being relevant in the immunity observed against the specific pathogen. The antibody variable genes will be cloned by RT-PCR, and inserted in pre-built vector scaffolds of constant regions with defined features (e.g. non-inflammatory, stable in mucus, valency, potential to perform effector functions). These huMabs will then be expressed as part of the Production Core in a transient plant system capable of producing milligram quantities in 1-2 weeks for in vitro and in vivo evaluation. In the second Specific Aim, huMabs will be evaluated in vitro for neutralization activity and some huMabs will be further characterized in mouse vaginal challenge models that have been established for evaluating microbicides and vaccines. Resulting data will help identify antibody correlates of protection from STIs (e.g. identification of protective epitopes, the role of antibody isotype, and the neutralizing titer of mucosal antibodies necessary for protection), providing guidance in vaccine development, and may yield huMabs worthy of clinical development as microbicides.